Many cytolytic proteins have been described (Lesieur et al. Mol. Membr. Biol. 14:45064, 1997). These naturally occurring cytotoxic proteins include mammalian proteins such as perforin, and bacterial proteins such as aerolysin (produced by Aeromonas hydrophila), α-hemolysin (produced by Staphylococcus aureus), alpha toxin (produced by Clostridium septicum), δ-toxin (produced by Bacillus thuringiensis), anthrax protective antigen, Vibrio cholerae VCC toxin, Staphylococcus leucocidins, LSL toxin from Laetiporus sulphureus, epsilon toxin from Clostridium perfringens, and hydralysins produced by Cnidaria spp.
Some of these cytotoxic proteins, for example, proaerolysin and alpha toxin, are synthesized as inactive protoxins. These protoxins contain discrete functionalities including a binding domain, which allows binding of the protoxin to a cell, a toxin domain, and either an N-terminal or a C-terminal inhibitory peptide domain that contains a protease cleavage site. Cleavage of the inhibitory peptide domain at the protease cleavage site results in activation of the protoxin, leading to oligomerization of the cytotoxin in the plasma membrane, producing pores that lead to rapid cytolytic cell death (Rossjohn et al. J. Struct. Biol. 121:92-100, 1998). Pore formation physically disrupts the cell membranes, and results in death of cells in all phases of the cell cycle, including non-proliferating cells (i.e. G0 arrested). These cytotoxins are not specific in the type of cells they are able to kill, as their binding domains target molecules that are found on most cells, and they are generally activated by proteases that are not cell-specific.
Cytolytic pore-forming proteins or modified versions of these proteins have been proposed as potential therapeutics for the treatment of cancer. For example, U.S. Pat. No. 5,777,078 describes pore-forming agents that are activated at the surface of a cell by a number of conditions, including proteolysis, to lyse the cell. These pore-forming agents can be used generally to destroy unwanted cells associated with a pathological condition in an animal. Such cells include but are not limited to tumor cells, cells which are chronically infected with virus, or cells, which when improperly regulated or expressed, result in a disease state, e.g., cells of the immune system. WO 98/020135 describes methods and compositions relating to Pseudomonas exotoxin proproteins modified for selective toxicity. The exotoxin is modified to be activated by a desired protease by insertion of a protease susceptible sequence in the proprotein. In one example the exotoxin is modified to insert a prostate specific antigen (PSA) cleavage site for the purpose of targeting and killing prostate cancer cells.
U.S. Patent Application No. 2004/0235095 describes the use of modified cytolytic pore-forming proteins for the treatment of prostate and other cancers. The cytolytic proteins can be modified to include a prostate-specific cleavage site, and/or a prostate-specific targeting domain and can be used to selectively target and kill prostate cancer cells.
Cancer is characterized by an increase in the number of abnormal, or neoplastic cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Unlike normal cells, in general, cancer cells continue to reproduce, they do not specialize or become mature, and they have the ability to spread from the tissue of origin to other locations within the body. These characteristics of cancer cells generally result from changes in the relative pattern of gene expression within these cells compared to that in normal cells. Many strategies for developing therapeutics for the treatment of cancer have focused on taking advantage of the differences in gene expression between normal cells and cancer cells, and targeting cancer cells using molecular markers that are specific to cancer cells.
In contrast, benign prostatic hyperplasia (BPH, also known as benign prostatic hypertrophy) is a non-cancerous condition resulting from enlargement of the prostate gland as a consequence of the natural progression of prostate growth with age. Enlargement of the prostate can be a result of increased prostate cell proliferation, or an increase in prostate cell size. This progressive prostate growth does not usually cause problems until late in life. The National Institute of Health (NIH) estimates that 60% of American men in their sixties have some symptoms of BPH and that the condition affects more than 90% of men in their seventies and eighties. Approximately 115 million males worldwide in the 50+ age group have varying degrees of BPH. Due to the aging of the population, the prevalence is expected to increase substantially over the next 20 years. Severe BPH can cause serious problems such as urinary tract infections, bladder and kidney damage, including bladder stones, incontinence and most seriously, gross hematuria and renal failure due to obstructive uropathy.
There are several strategies currently available for treating BPH. These include watchful waiting, medical therapy such as alpha blocker therapy and finasteride therapy, balloon dilation and various surgical procedures such as transurethral incision of the prostate (TUIP), transurethral resection of the prostate (TURP), and open prostatectomy. Few treatments are without any adverse consequences, and this is particularly so with treatments for BPH, where there is a delicate balancing act between the benefits and demerits of the treatments available. The adverse events following currently available treatments for BPH include impotence (for various surgical procedures ranging from about 4% to 40%, the incidence of impotence is also increased after some medical treatments), incontinence (stress incontinence about 3% after surgery, with total urinary incontinence approaching 1%), and the need for re-treatment. Combined analysis of published data estimated that the mean probability for perioperative mortality (death within 90 days of a procedure) was 1.5% for TURP. For open surgery it was 2.4% and for balloon dilation it was 3.5%.
Currently, the most commonly used hormone therapy is oral administration of finasteride. Finasteride, commercially available under the tradename Proscar™ from Merck & Co. Inc., Whitehouse Station, N.J., is a synthetic 4-azasteroid compound, a specific inhibitor of steroid Type II 5α-reductase, and an intracellular enzyme that converts the androgen testosterone into 5α-dihydrotestosterone (DHT). Finasteride helps to shrink the enlarged prostate and reduces elevated PSA due to benign prostate conditions. However, finasteride is known to cause undesirable side effects, which include impotence or lessened desire for sex, problems with ejaculation, and breast enlargement and/or tenderness. Dutasteride (Duagen) is another drug for the treatment of BPH and it is capable of blocking both types I and II 5α-reductase. Sexual side effects are similar to those of finasteride.
Alpha-1 adrenoceptor blocking agents are also currently used for clinical treatment of benign prostatic hyperplasia. Examples include tamsulasin hydrochloride, terazosin hydrochloride, alfuzosin hydrochloride and doxazosin mesylate. The reduction in symptoms of BPH and improvement in urine flow rates following administration of an alpha-1 adrenoceptor blocking agent are related to relaxation of smooth muscle produced by blockage of alpha-1 adrenoceptors in the bladder neck and prostate.
Furthermore, plant sterols and extracts have also been used for the treatment of benign prostatic hyperplasia.
United States Patent Application No. 20040081659 describes conjugates useful to treat BPH comprising 1) oligopeptides with amino acid sequences which are selectively and proteolytically cleaved by PSA, chemically linked to 2) vinca alkaloid cytotoxic agents. Theoretically, the cytotoxic activity of the alkaloid is low in the conjugate and increased when the linkage is cleaved by PSA.
European Patent Application 0652014 describes a treatment for BPH comprising administration of PSA (prostate-specific antigen) linked to an immunogenic carrier to induce the production of anti-PSA antibodies. Anti-PSA antibodies may also be used. The immunogenic carrier can be tetanus toxin, diphtheria toxin or cholera toxin chain B.
U.S. Pat. No. 6,379,669 describes a method of targeting a specific organ by coupling a therapeutic agent to an antibody or fragments thereof. Such coupled therapeutic agents (or immunoconjugates) can be used to treat prostate cancer, BPH, or prostatitis. The immunoconjugates included are antibodies against PSA that are linked to various bioactive agents. The bioactive agents may include bacterial toxins. Similarly, in United States Patent Application No. 20020001588, the chemical linkage of antibodies and various bioactive therapeutic agents is explored further.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.